The large-conductance, Ca2+-activated K+ channel from cardiac mitochondria (mitoBKCa) is thought to play a role in cardioprotection. MitoBKCa molecular size is uncertain with reported immunochemical signals at ~55 and ~125 kDa. In addition, mitoBKCa molecular identity and its mitochondrial targeting mechanisms remain unknown, while there is scarce information about its functional properties or direct evidence for their role in cardioprotection. Because cardiac mitoBKCa shares conductance, Ca2+ responsiveness, and sensitivity to pharmacological agents with its plasma membrane counterpart known as BKCa (or MaxiK), we expect that mitoBKCa is assembled like BKCa by four pore-forming a subunits with a monomeric mass of ~125 kDa. We will now test the hypotheses that: 1) mitoBKCa and plasma membrane BKCa are encoded by the same gene and splice variation provides BKCa with intrinsic signals for its preferential mitochondrial targeting; 2) the normal absence of BKCa from the cardiomyocyte plasmalemma and presence in mitochondria is ruled by both an intrinsic signal(s) within mitoBKCa backbone (i.e. splice insert) either directly or indirectly (i.e. via a chaperone), and by cell-specific mechanisms, and ) mitoBKCa contributes to cardioprotection by regulating mitochondrial calcium retention capacity (CRC) and permeability transition pore (mPTP) opening. Preliminary Data shows: 1) the detection of a ~125 kDa protein in mitochondria by specific anti-BKCa antibodies; 2) the detection of all 27 constitutive BKCa exons in isolated cardiomyocyte mRNAs; 3) that BKCa isoform containing splice insert DEC (C-terminal insert of 61 amino acids) but not the constitutive form of BKCa (insertless BKCa) is readily targeted to mitochondria in adult cardiomyocytes; 4) that mitoBKCa subproteome uncovered as a partner Hsp60, a heat shock protein relevant for folding of mitochondrial imported proteins; and 5) that BKCa gene ablation prevents the cardioprotective action of putative BKCa channel opener NS1619. Overall the data support the above hypotheses, which will be tested using multiple approaches and pursuing the following Specific Aims to: 1. Identify the molecular correlate of cardiac mitoBKCa; 2. Functionally validate the identity of cloned putative mitoBKCa; 3. Determine signal mechanisms involved in mitoBKCa mitochondrial targeting; and 4. Directly address the role of mitoBKCa in cardioprotection. The outcomes of this program will open the opportunity to study mitoBKCa at the molecular level and advance the cardiac field by: solving mitoBKCa identity, providing information on its targeting mechanisms, and defining its functional properties and role in cardioprotection. Further understanding of the underlying molecular mechanism(s) of mitoBKCa cardioprotective effects will provide new targets for translation into therapeutics.